Pim complete

Cells were harvested in ice cold PBS and lysed in RIPA buffer (Pierce) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, total protein in the cell extracts was quantified using Rotiquant (Carl Roth). Forty micrograms of protein was resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-caspase 3, anti-beta actin (Cell Signaling), or anti COX4 (Santa cruz) primary antibodies followed by secondary horse-radish peroxidase-coupled antibodies (Rockland Immunochemicals), and signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.

Following the indicated incubation, cells were harvested in ice-cold PBS and lysed in RIPA buffer (ThermoFisher, Waltham, MA, USA) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche, Basal, Switzerland) for 20 min on ice. After centrifugation at 15,000× g for 15 min at 4 °C, the total protein in the whole-cell extracts was quantified using Rotiquant (Carl Roth, Karlsruhe, Germany). Twenty-five micrograms of protein were resolved by SDS-PAGE and blotted on polyvinylidene fluoride membranes (both Invitrogen, Carlsbad, CA, USA). The membranes were probed with primary anti-p53 and anti-β-actin antibodies, followed by anti-rabbit antibodies coupled to horseradish peroxidase (all Santa Cruz, Dallas, TX, USA). Signals were acquired in a chemiluminescence detection system (Applied Biosystems, Foster City, CA, USA) in a linear dynamic range mode.

Cells were harvested in ice-cold phosphate-buffered saline (PBS). Harvested cells were lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). In all, 40 µg of protein was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen) and blotted on polyvinylidene fluoride membranes (Invitrogen). The membranes were probed with anti-OCT6 or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase-coupled antibodies (Rockland Immunochemicals), and signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.